Electrodeposited nickel-iron nanoclusters and nickel-iron-graphene oxide onto a carbon electrode was fabricated to enhance charge transfer while at the same time permitting adsorption of organic molecules to the carbon surface. Ni–Fe and Ni–Fe–graphene oxide were prepared from a sulfate-boric acid and sulfate-boric acid-graphene oxide water dispersion electrolyte at a pH of 3 by a pulse galvanostatic method onto a glassy carbon disk electrode. Electrochemical impedance spectroscopy (EIS) was conducted on the Ni–Fe/glassy carbon and Ni–Fe–graphene oxide/glassy carbon composite electrodes in a phosphate buffer solution (PBS) containing various concentrations of methylene blue (MB) or pyocyanin (PYO). MB is used as a dye and is a pollutant, and can serve as a comparable model for PYO due to similar structure and redox potential. PYO is a metabolite of the Pseudomonas aeruginosa, a gram-negative bacterium, associated with disease in humans responsible for chronic infection in people with cystic fibrosis and in burn patients; thus its detection is of interest. The R_sC parameters determined from EIS equivalent circuit models for the interfacial region of the Ni–Fe/glassy carbon electrode with an aqueous electrolyte containing MB or PYO had a larger signal as the analyte concentration decreased, in contrast to conventional voltammetry techniques. The fractional adsorption with concentration for MB followed a Langmuir adsorption isotherm, the adsorption energy determined, and compared with PYO. The electrode fabricated with graphene oxide in the electrolyte increased the R_sC value under PYO detection.